Reliable enumeration of malaria parasites in thick blood films using digital image analysis

<p>Abstract</p> <p>Background</p> <p>Quantitation of malaria parasite density is an important component of laboratory diagnosis of malaria. Microscopy of Giemsa-stained thick blood films is the conventional method for parasite enumeration. Accurate and reproducible parasite counts are difficult to achieve, because of inherent technical limitations and human inconsistency. Inaccurate parasite density estimation may have adverse clinical and therapeutic implications for patients, and for endpoints of clinical trials of anti-malarial vaccines or drugs. Digital image analysis provides an opportunity to improve performance of parasite density quantitation.</p> <p>Methods</p> <p>Accurate manual parasite counts were done on 497 images of a range of thick blood films with varying densities of malaria parasites, to establish a uniformly reliable standard against which to assess the digital technique. By utilizing descriptive statistical parameters of parasite size frequency distributions, particle counting algorithms of the digital image analysis programme were semi-automatically adapted to variations in parasite size, shape and staining characteristics, to produce optimum signal/noise ratios.</p> <p>Results</p> <p>A reliable counting process was developed that requires no operator decisions that might bias the outcome. Digital counts were highly correlated with manual counts for medium to high parasite densities, and slightly less well correlated with conventional counts. At low densities (fewer than 6 parasites per analysed image) signal/noise ratios were compromised and correlation between digital and manual counts was poor. Conventional counts were consistently lower than both digital and manual counts.</p> <p>Conclusion</p> <p>Using open-access software and avoiding custom programming or any special operator intervention, accurate digital counts were obtained, particularly at high parasite densities that are difficult to count conventionally. The technique is potentially useful for laboratories that routinely perform malaria parasite enumeration. The requirements of a digital microscope camera, personal computer and good quality staining of slides are potentially reasonably easy to meet.</p

Inter-rater reliability of malaria parasite counts and comparison of methods

BACKGROUND: The introduction of artemesinin-based treatment for falciparum malaria has led to a shift away from symptom-based diagnosis. Diagnosis may be achieved by using rapid non-microscopic diagnostic tests (RDTs), of which there are many available. Light microscopy, however, has a central role in parasite identification and quantification and remains the main method of parasite-based diagnosis in clinic and hospital settings and is necessary for monitoring the accuracy of RDTs. The World Health Organization has prepared a proficiency testing panel containing a range of malaria-positive blood samples of known parasitaemia, to be used for the assessment of commercially available malaria RDTs. Different blood film and counting methods may be used for this purpose, which raises questions regarding accuracy and reproducibility. A comparison was made of the established methods for parasitaemia estimation to determine which would give the least inter-rater and inter-method variation METHODS: Experienced malaria microscopists counted asexual parasitaemia on different slides using three methods; the thin film method using the total erythrocyte count, the thick film method using the total white cell count and the Earle and Perez method. All the slides were stained using Giemsa pH 7.2. Analysis of variance (ANOVA) models were used to find the inter-rater reliability for the different methods. The paired t-test was used to assess any systematic bias between the two methods, and a regression analysis was used to see if there was a changing bias with parasite count level. RESULTS: The thin blood film gave parasite counts around 30% higher than those obtained by the thick film and Earle and Perez methods, but exhibited a loss of sensitivity with low parasitaemia. The thick film and Earle and Perez methods showed little or no bias in counts between the two methods, however, estimated inter-rater reliability was slightly better for the thick film method. CONCLUSION: The thin film method gave results closer to the true parasite count but is not feasible at a parasitaemia below 500 parasites per microlitre. The thick film method was both reproducible and practical for this project. The determination of malarial parasitaemia must be applied by skilled operators using standardized techniques

Malaria vaccine efficacy: the difficulty of detecting and diagnosing malaria

New sources of funding have revitalized efforts to control malaria. An effective vaccine would be a tremendous asset in the fight against this devastating disease and increasing financial and scientific resources are being invested to develop one. A few candidates have been tested in Phase I and II clinical trials, and several others are poised to begin trials soon. Some studies have been promising, and others disappointing. It is difficult to compare the results of these clinical trials; even independent trials of the same vaccine give highly discrepant results. One major obstacle in evaluating malaria vaccines is the difficulty of diagnosing clinical malaria. This analysis evaluates the impact of diagnostic error, particularly that introduced by microscopy, on the outcome of efficacy trials of malaria vaccines and make recommendations for improving future trials

The Impact of IPTi and IPTc Interventions on Malaria Clinical Burden – In Silico Perspectives

Background: Clinical management of malaria is a major health issue in sub-Saharan Africa. New strategies based on intermittent preventive treatment (IPT) can tackle disease burden by simultaneously reducing frequency of infections and life-threatening illness in infants (IPTi) and children (IPTc), while allowing for immunity to build up. However, concerns as to whether immunity develops efficiently in treated individuals, and whether there is a rebound effect after treatment is halted, have made it imperative to define the effects that IPTi and IPTc exert on the clinical malaria scenario. Methods and Findings: Here, we simulate several schemes of intervention under different transmission settings, while varying immunity build up assumptions. Our model predicts that infection risk and effectiveness of acquisition of clinical immunity under prophylactic effect are associated to intervention impact during treatment and follow-up periods. These effects vary across regions of different endemicity and are highly correlated with the interplay between the timing of interventions in age and the age dependent risk of acquiring an infection. However, even when significant rebound effects are predicted to occur, the overall intervention impact is positive. Conclusions: IPTi is predicted to have minimal impact on the acquisition of clinical immunity, since it does not interfere with the occurrence of mild infections, thus failing to reduce the underlying force of infection. On the contrary, IPTc has

Use of remote sensing to identify spatial risk factors for malaria in a region of declining transmission: a cross-sectional and longitudinal community survey

<p>Abstract</p> <p>Background</p> <p>The burden of malaria has decreased dramatically within the past several years in parts of sub-Saharan Africa. Further malaria control will require targeted control strategies based on evidence of risk. The objective of this study was to identify environmental risk factors for malaria transmission using remote sensing technologies to guide malaria control interventions in a region of declining burden of malaria.</p> <p>Methods</p> <p>Satellite images were used to construct a sampling frame for the random selection of households enrolled in prospective longitudinal and cross-sectional surveys of malaria parasitaemia in Southern Province, Zambia. A digital elevation model (DEM) was derived from the Shuttle Radar Topography Mission version 3 DEM and used for landscape characterization, including landforms, elevation, aspect, slope, topographic wetness, topographic position index and hydrological models of stream networks.</p> <p>Results</p> <p>A total of 768 individuals from 128 randomly selected households were enrolled over 21 months, from the end of the rainy season in April 2007 through December 2008. Of the 768 individuals tested, 117 (15.2%) were positive by malaria rapid diagnostic test (RDT). Individuals residing within 3.75 km of a third order stream were at increased risk of malaria. Households at elevations above the baseline elevation for the region were at decreasing risk of having RDT-positive residents. Households where new infections occurred were overlaid on a risk map of RDT positive households and incident infections were more likely to be located in high-risk areas derived from prevalence data. Based on the spatial risk map, targeting households in the top 80^thpercentile of malaria risk would require malaria control interventions directed to only 24% of the households.</p> <p>Conclusions</p> <p>Remote sensing technologies can be used to target malaria control interventions in a region of declining malaria transmission in southern Zambia, enabling a more efficient use of resources for malaria elimination.</p

Declining Burden of Malaria Over two Decades in a Rural Community of Muheza District, North-Eastern Tanzania.

The recently reported declining burden of malaria in some African countries has been attributed to scaling-up of different interventions although in some areas, these changes started before implementation of major interventions. This study assessed the long-term trends of malaria burden for 20 years (1992--2012) in Magoda and for 15 years in Mpapayu village of Muheza district, north-eastern Tanzania, in relation to different interventions as well as changing national malaria control policies.\ud Repeated cross-sectional surveys recruited individuals aged 0 -- 19 years from the two villages whereby blood smears were collected for detection of malaria parasites by microscopy. Prevalence of Plasmodium falciparum infections and other indices of malaria burden (prevalence of anaemia, splenomegaly and gametocytes) were compared across the years and between the study villages. Major interventions deployed including mobile clinic, bed nets and other research activities, and changes in national malaria control policies were also marked. In Magoda, the prevalence of P. falciparum infections initially decreased between 1992 and 1996 (from 83.5 to 62.0%), stabilized between 1996 and 1997, and further declined to 34.4% in 2004. A temporary increase between 2004 and 2008 was followed by a progressive decline to 7.2% in 2012, which is more than 10-fold decrease since 1992. In Mpapayu (from 1998), the highest prevalence was 81.5% in 1999 and it decreased to 25% in 2004. After a slight increase in 2008, a steady decline followed, reaching <5% from 2011 onwards. Bed net usage was high in both villages from 1999 to 2004 (>=88%) but it decreased between 2008 and 2012 (range, 28% - 68%). After adjusting for the effects of bed nets, age, fever and year of study, the risk of P. falciparum infections decreased significantly by >=97% in both villages between 1999 and 2012 (p < 0.001). The prevalence of splenomegaly (>40% to <1%) and gametocytes (23% to <1%) also decreased in both villages.Discussion and conclusionsA remarkable decline in the burden of malaria occurred between 1992 and 2012 and the initial decline (1992 -- 2004) was most likely due to deployment of interventions, such as bed nets, and better services through research activities. Apart from changes of drug policies, the steady decline observed from 2008 occurred when bed net coverage was low suggesting that other factors contributed to the most recent pattern. These results suggest that continued monitoring is required to determine causes of the changing malaria epidemiology and also to monitor the progress towards maintaining low malaria transmission and reaching related millennium development goals

Webuye health and demographic surveillance systems baseline survey of soil-transmitted helmints and intestinal protozoa among children up to five years

Background. The intestinal parasitic infections (IPIs) are globally endemic, and they constitute the greatest cause of illness and disease worldwide. Transmission of IPIs occurs as a result of inadequate sanitation, inaccessibility to potable water, and poor living conditions. Objectives. To determine a baseline prevalence of IPIs among children of five years and below at Webuye Health and Demographic Surveillance (HDSS) area in western Kenya. Methods. Cross-sectional survey was used to collect data. Direct saline and formal-ether-sedimentation techniques were used to process the specimens. Descriptive and inferential statistics such as Chi-square statistics were used to analyze the data. Results. A prevalence of 52.3% (417/797) was obtained with the male child slightly more infected than the female (53.5% versus 51%), but this was not significant (χ2=0.482, P>0.05). Giardia lamblia and Entamoeba histolytica were the most common pathogenic IPIs with a prevalence of 26.1% (208/797) and 11.2% (89/797), respectively. Soil-transmitted helminths (STHs) were less common with a prevalence of 4.8% (38/797), 3.8% (30/797), and 0.13% (1/797) for Ascaris lumbricoides, hookworms, and Trichuris trichiura, respectively. Conclusions. Giardia lamblia and E. histolytica were the most prevalent pathogenic intestinal protozoa, while STHs were less common. Community-based health promotion techniques are recommended for controlling these parasites

Prevalence and Predictors of Urinary Tract Infection and Severe Malaria Among Febrile Children Attending Makongoro Health Centre in Mwanza City, North-Western Tanzania.

In malaria endemic areas, fever has been used as an entry point for presumptive treatment of malaria. At present, the decrease in malaria transmission in Africa implies an increase in febrile illnesses related to other causes among underfives. Moreover, it is estimated that more than half of the children presenting with fever to public clinics in Africa do not have a malaria infection. Thus, for a better management of all febrile illnesses among under-fives, it becomes relevant to understand the underlying aetiology of the illness. The present study was conducted to determine the relative prevalence and predictors of P. falciparum malaria, urinary tract infections and bacteremia among under-fives presenting with a febrile illness at the Makongoro Primary Health Centre, North-Western Tanzania. From February to June 2011, a cross-sectional analytical survey was conducted among febrile children less than five years of age. Demographic and clinical data were collected using a standardized pre-tested questionnaire. Blood and urine culture was done, followed by the identification of isolates using in-house biochemical methods. Susceptibility patterns to commonly used antibiotics were investigated using the disc diffusion method. Giemsa stained thin and thick blood smears were examined for any malaria parasites stages. A total of 231 febrile under-fives were enrolled in the study. Of all the children, 20.3% (47/231, 95%CI, 15.10-25.48), 9.5% (22/231, 95%CI, 5.72-13.28) and 7.4% (17/231, 95%CI, 4.00-10.8) had urinary tract infections, P. falciparum malaria and bacteremia respectively. In general, 11.5% (10/87, 95%CI, 8.10-14.90) of the children had two infections and only one child had all three infections. Predictors of urinary tract infections (UTI) were dysuria (OR = 12.51, 95% CI, 4.28-36.57, P < 0.001) and body temperature (40-41 C) (OR = 12.54, 95% CI, 4.28-36.73, P < 0.001). Predictors of P. falciparum severe malaria were pallor (OR = 4.66 95%CI, 1.21-17.8, P = 0.025) and convulsion (OR = 102, 95% CI, 10-996, P = 0.001). Escherichia coli were the common gram negative isolates from urine (72.3%, 95% CI, 66.50-78.10) and blood (40%, 95%CI, and 33.70-46.30). Escherichia coli from urine were 100% resistant to ampicillin, 97% resistant to co-trimoxazole, 85% resistant to augmentin and 32.4% resistant to gentamicin; and they were 100%, 91.2% and 73.5% sensitive to meropenem, ciprofloxacin and ceftriaxone respectively. Urinary tract infection caused by multi drug resistant Escherichia coli was the common cause of febrile illness in our setting. Improvement of malaria diagnosis and its differential diagnosis from other causes of febrile illnesses may provide effective management of febrile illnesses among children in Tanzania

Worldwide Incidence of Malaria in 2009: Estimates, Time Trends, and a Critique of Methods

Richard Cibulskis and colleagues present estimates of the worldwide incidence of malaria in 2009, together with a critique of different estimation methods, including those based on risk maps constructed from surveys of parasite prevalence, and those based on routine case reports compiled by health ministries

Accuracy of Malaria Rapid Diagnostic Tests in Community Studies and their Impact on Treatment of Malaria in an Area with Declining Malaria Burden in North-Eastern Tanzania.

Despite some problems related to accuracy and applicability of malaria rapid diagnostic tests (RDTs), they are currently the best option in areas with limited laboratory services for improving case management through parasitological diagnosis and reducing over-treatment. This study was conducted in areas with declining malaria burden to assess; 1) the accuracy of RDTs when used at different community settings, 2) the impact of using RDTs on anti-malarial dispensing by community-owned resource persons (CORPs) and 3) adherence of CORPs to treatment guidelines by providing treatment based on RDT results. Data were obtained from: 1) a longitudinal study of passive case detection of fevers using CORPs in six villages in Korogwe; and 2) cross-sectional surveys (CSS) in six villages of Korogwe and Muheza districts, north-eastern, Tanzania. Performance of RDTs was compared with microscopy as a gold standard, and factors affecting their accuracy were explored using a multivariate logistic regression model. Overall sensitivity and specificity of RDTs in the longitudinal study (of 23,793 febrile cases; 18,154 with microscopy and RDTs results) were 88.6% and 88.2%, respectively. In the CSS, the sensitivity was significantly lower (63.4%; χ2=367.7, p<0.001), while the specificity was significantly higher (94.3%; χ2=143.1, p<0.001) when compared to the longitudinal study. As determinants of sensitivity of RDTs in both studies, parasite density of<200 asexual parasites/μl was significantly associated with high risk of false negative RDTs (OR≥16.60, p<0.001), while the risk of false negative test was significantly lower among cases with fever (axillary temperature ≥37.5 °C) (OR≤0.63, p≤0.027). The risk of false positive RDT (as a determinant of specificity) was significantly higher in cases with fever compared to afebrile cases (OR≥2.40, p<0.001). Using RDTs reduced anti-malarials dispensing from 98.9% to 32.1% in cases aged ≥5 years. Although RDTs had low sensitivity and specificity, which varied widely depending on fever and parasite density, using RDTs reduced over-treatment with anti-malarials significantly. Thus, with declining malaria prevalence, RDTs will potentially identify majority of febrile cases with parasites and lead to improved management of malaria and non-malaria fevers